Molecular epidemiology, transmission and clinical features of 2022‐mpox outbreak: A systematic review

Abstract Background and Aims The 2022‐mpox outbreak has spread worldwide in a short time. Integrated knowledge of the epidemiology, clinical characteristics, and transmission of mpox are limited. This systematic review of peer‐reviewed articles and gray literature was conducted to shed light on the epidemiology, clinical features, and transmission of 2022‐mpox outbreak. Methods We identified 45 peer‐reviewed manuscripts for data analysis. The standards of the Preferred Reporting Items for Systematic Review and Meta‐Analysis (PRISMA) Statement and Cochrane Collaboration were followed for conducting the study. Results The case number of mpox has increased about 100 times worldwide. About 99% of the cases in 2022 outbreak was from non‐endemic regions. Men (70%–98% cases) were mostly infected with homosexual and bisexual behavior (30%–60%). The ages of the infected people ranged between 30 and 40 years. The presence of HIV and sexually transmitted infections among 30%–60% of cases were reported. Human‐to‐human transmission via direct contact and different body fluids were involved in the majority of the cases (90%–100%). Lesions in genitals, perianal, and anogenital areas were more prevalent. Unusually, pharyngitis (15%–40%) and proctitis (20%–40%) were more common during 2022 outbreak than pre‐2022 outbreaks. Brincidofovir is approved for the treatment of smallpox by FDA (USA). Two vaccines, including JYNNEOSTM and ACAM2000®, are approved and used for pre‐ and post‐prophylaxis in cases. About 100% of the cases in non‐endemic regions were associated with isolates of IIb clade with a divergence of 0.0018–0.0035. Isolates from B.1 lineage were the most predominant followed by B.1.2 and B.1.10. Conclusion This study will add integrated knowledge of the epidemiology, clinical features, and transmission of mpox.


| INTRODUCTION
The 2022 mpox (previously monkeypox) outbreak has transmitted across 110 countries with case number surpassing 86,716 on March 29, 2023. 1,24][5] During 1970s, several outbreaks were reported from monkeys in the United States and the Netherlands.[5] Before the ongoing 2022 outbreak, mpox was considered a rare zoonotic disease. 4Monkeypox virus is a double-stranded DNA virus belonging to the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus. 6The genus includes other important pathogens of humans and animals, including mpox, cowpox, camelpox, vaccinia, and the smallpox.Symptomatic infection of monkeypox virus results in smallpox-like symptoms in patients.Further, monkeypox and smallpox viruses are highly similar both in genetic and antigenic properties. 6,7The genome of monkeypox virus is about 200 kb in size, linear, and contains hairpin ends with inverted terminal repeats.Nearly 200 proteins are encoded by mpox virus.Housekeeping genes are encoded from the central conserved regions.Antigenic proteins are encoded by the terminal regions of the genome and vary between poxviruses. 6,7e animal reservoirs of zoonotic monkeypox virus are still unknown.Further, the intermediate hosts of monkeypox virus are also unknown. 4,6,7Apes and monkeys are the most likely intermediate hosts and several rodent species, including tree squirrels, rope squirrels, Gambian pouched rats, and dormice, are the most probable animal reservoir of monkeypox virus. 4The natural history of monkeypox virus is yet to be discovered.
6][17][18][19][20][21] Parenteral transmission and fetal deaths of mpox have been reported. 21,22tients with monkeypox virus infection develop characteristic symptoms. 23,248][29] Severe illnesses and other complications like encephalitis, pneumonitis, and secondary infections are higher in children, elderly, and HIV-infected patients. 23,25,27,29e discontinuation of routine vaccination of smallpox since 1980 and asymptomatic circulation of monkeypox virus in humans may have contributed significantly to changes of biological properties of the virus. 4,6,7Further, changes in human behaviors and movement have probably contributed to the 2022 outbreaks.Studies on the epidemiology, clinical characteristics, and transmission route are scarce.Only few review studies are available with limited knowledge of the previous sporadic outbreaks. 4,6,7Recently, as case numbers are increasing, studies focusing on clinical features and transmission are getting highlighted to understand the baseline of the outbreaks.This study was conducted to create integrated insights about the epidemiology, route of transmission in the human body and environment along with clinical data of monkeypox virus.

| Definitions
The epidemiology of mpox is defined as the distribution and determinants of outbreaks in different populations and strategies taken to minimize the health effect in different population.The clinical feature was defined, including both the signs and symptoms during and after the mpox infection.Transmission of monkeypox virus was defined as the transfer of the virus from human to human, reservoirs to human, and carrier to human.This study included epidemiological, clinical, and virological studies.The PCR-positive laboratory-confirmed test was defined as the positive mpox case.The standards of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement and Cochrane Collaboration were followed for conducting the study. 30The study is submitted for registration in the PROSPERO (ID 391380).

| Study design
The study was conducted by the following different steps, including identification of precise objectives and search strategies, appropriate research articles, inclusion of manuscripts, collection of data, analysis, and summarization of the findings.This study included previous findings from epidemiological studies, case studies, outbreak investigation, surveillance work, and online databases.No strict parameters for the quality assessment of these studies are available.As a result, this study relied on the quality report of the selected articles by the authors.

| Search strategy and selection criteria
We performed searches for articles in MEDLINE (through PubMed), EMBASE, Web of Science, Scopus, the Internet Library sub-Saharan Africa (ilissAfrica), African Journals Online (AJOL), The New England Journal of Medicine (NEJM) and The Lancet with no restriction on duplicated articles, and removing correspondence or comment of duplicated data were performed by N. S., S. K. D., and K. A. J. separately.
The seasonal exclusion criteria could not be implemented due to lack of studies on seasonality and environmental impacts.Furthermore, we could not exclude studies that did not include data on specific clades due to a lack of work.We used the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) assessment tool for measuring the risk of bias. 31,32The SYRCLE consists of 10 parameters to assess different biases in studies.The parameters included detection bias, attrition bias, reporting bias, selection bias, performance bias, and other biases.The bias for each parameter was measured by using the possible outcomes as yes, no, and unclear, representing low, high, and unclear bias, respectively. 31,32

| Case definition
A confirmed case of monkeypox virus was defined by following the UK Health Security Agency (UKHSA) definition: a positive result on mpox PCR assay in a specimen collected from any anatomical site is considered as a positive case.The type of the used PCR assay will be determined on the basis of local guidelines and availability of the test method.

| Statistical analysis
A total number of cases was determined for each decade by summation of the reported cases per clade.Pooled Statistical analyses were conducted by using SAS version 9.4.

| Studies included
This study detected 12,032 research articles on epidemiology and transmission of monkeypox virus and previously mentioned related search terms (Figure 1).Among 12,032 articles, 453 articles were screened and considered eligible for further full-text investigation.
The excluded articles were duplicate, reviews, correspondence, editorial, and failed to have inclusion criteria.After analyzing the full texts, only 66 (14.5%, 66 of 453) studies were found to be eligible for further analysis.Based on the inclusion criteria, 45 of 66 (68%) manuscripts were finally selected.Among the selected manuscripts, we extracted epidemiological data from 30 articles, transmission data from 33 articles, clinical symptoms from 18 articles, and treatment and prevention from 9 articles.We also extracted data from two databases, including CDC (USA) and ECDC (Europe) (Figure 1).
21,23,24,[26][27][28][29] The epidemiological curve of the first wave of mpox pandemic has been visualized.One peak with the highest number of confirmed cases reaching 1000 cases/day was apparently confined within a period of 4 months, July 2022 to October 2022.2][3] Further, in mode of transmission we have noticed majority of the study identified involvement of sexual activities in cases of human-to-human transmission (Table 1).Another significant unusual characteristic was the higher frequency (60%-100%) of cases identified among men who have sex with men (MSM) and HIV patients.  As e pandemic is progressing more studies are required to characterize the epidemiology.

| Clade distribution of monkeypox virus
2][53] In the phylogenetic analysis of monkeypox virus, it was documented that isolates from clade IIb were associated with 2022-outbreak.The tree included 700 genome and MPXV-M5312_HM12_Rivers was used as the reference sequence.
[55] Data from about 1400 whole genome were included for hmpxv1.
All of the whole-genome sequences of hmpxv1 are from the clade IIa.3][54][55] The isolates of B.1 lineage and their descendants have evolved and acquired successful human-to-human transmission capability (Table 2).Though the natural history of 2022 mpox pandemic is not well understood, it has several characteristic features than the previous mpox endemic cases, outbreaks, and sporadic cases (Table 1).The most notable epidemiological features of newly evolved isolates include a defined risk group, namely MSM, sexual transmission, and increased median age than previous outbreaks.The pandemic lineages have probably undergone genetic evolution before the onset of the first case of the ongoing pandemic in the first quarter of 2022 and acquired more changes in the genome through human-to-human transmission.
Extensive studies are required to determine the relationship of evolutionary changes with the newly identified pandemic characteristics.
SHARIF ET AL.

| Transmission of mpox virus during outbreaks
Transmission of mpox virus has been occurring since 1960s in the nonhuman primates.[59][60] The natural history of mpox virus is yet to be understood well.In the endemic areas, the zoonotic transmission was the most prominent source of human infection (Table 3).However, over time the regions of Africa (Table 3).The growth (R 0 ) was estimated below 1 both for the clade I and clade II in endemic and pandemic cases.
Traveler cases were mostly reported from clade II and the virus was carried outside the endemic areas.Limited evidence on transmission chains support smaller clusters (not more than 10) of infection from the indexed patient can occur.
As the ongoing outbreak involves the largest number of human cases, the knowledge of the diversity of transmission continues to evolve.[27][28][29] Transmission of monkeypox virus in people after sexual activities with presymptomatic patients has been documented.However, transmission from asymptomatic patients is not reported yet.Virological data on several studies have documented infectious monkeypox virus in semen in patients.
[27][28][29] Several studies have reported that isolated DNA of monkeypox virus from the lesions of anogenital skin and semen has the capability to infect cell lines like Vero E6 cells and produce cytopathic effects. 12,13,15,17,19,24,26,27However, these are preliminary studies to define and characterize the transmission of monkeypox virus via semen.We have also analyzed several studies reporting early evidence of droplet transmission of monkeypox virus via the respiratory route.Nosocomial infection among the healthcare providers has been documented.56,58,[60][61][62] There is limited evidence on the fomite transmission of monkeypox virus via contaminated surfaces and objects.However, the potential of contamination of different household objects by monkeypox virus should be evaluated in detail.
Studies have documented the presence of infectious monkeypox virus on lesions and major secretions, including urine, feces, nasal or oral droplets and conjunctival exudates in humans and animals. 21,23,28,29,50,56,58,60During 2022-outbreak the probable route of transmission included direct contact, ingestion, and inhalation.Pre-2022 mpox outbreaks and sporadic cases have transmitted to humans via bites from infected animals, contact with lesions or fluids and blood of infected organs, and aerosols. 4,50,56,58,60No confirmed sexual transmission was reported before 2022 outbreak. 4Both animal and person-to-person transmission were reported from pre-2022 outbreaks.However, during 2022 outbreak person-to-person transmission became the major source of transmission.It is unlikely that person-to-person transmission can maintain the virus in humans for a longer period.
4][65][66] The full host range of monkeypox virus is unknown.Documented cases of monkeypox virus include different animals like Old and New World T A B L E 2 Distribution of clades of monkeypox virus along with major epidemiological features.T A B L E 3 Host range, transmission history to humans, and isolation source of mpox virus.
3][74][75][76] Recently, it has been used to treat patients with monkeypox virus.The specific effects of cidofovir on the outcome of monkeypox virus need to be studied in detail.Cidofovir is also not approved for use in treating mpox by FDA (USA) or CDC.Brincidofovir is an oral formulation of cidofovir.It is already approved for the treatment of smallpox by FDA (USA).However, a limited number of studies have reported the effectiveness of brincidofovir against mpox infection.8][79] However, the specific effectiveness and side effects of these antivirals in mpox patients need to be evaluated in more detail.
Termination of smallpox vaccination contributed toward the uprise of monkeypox virus cases.4][75] The smallpox vaccine provides good cross-protection against mpox infection.For the prevention of mpox, vaccines, including JYNNEOSTM and ACAM2000 ® , are approved and used for pre-and post-prophylaxis in specific patients.JYNNEOSTM is a 2-dose vaccine. 75For vaccinations with JYNNEOSTM, a list of recommendation from CDC, USA should be followed.[75]

| DISCUSSION
2][3] This systematic review integrated epidemiological, clinical, and transmission data of 2022 outbreak of mpox.We used a structured format of data analysis and provided a real-world overview of mpox outbreak.2][3] This is one of the first systematic reviews reporting human-to-human transmission of monkeypox virus involving about 90,000 people in 110 regions.In mpox history, it is the largest known outbreak.Local transmission of mpox involving large number of cases in non-endemic regions has never been documented before 2022 outbreak.[3]5 The highest number of cases was reported in the Americas (nearly 70%) and EU/EEA (26%) regions, which is the first time in the history of monkeypox virus.The majority of the cases have been documented in the United States (30,000), followed by Brazil (10,599), Spain (7505), France (4114), and Colombia (4049), respectively.
2][3][4] There are several reasons behind this dramatic outbreak of monkeypox virus in the nonendemic regions.First of all, waning of mass immunity against smallpox has contributed toward the larger outbreak of monkeypox virus.Vaccines against smallpox are effective in providing protection against monkeypox virus.Second, genetic changes and evolution of monkeypox virus may have contributed to the changing epidemiology.41,46 This 2022-mpox outbreak may be a sign of a larger pandemic of mopox in the future.Without proper investigation and epidemiological analysis, it will be difficult to prevent mpox outbreaks in the future.
Majority of the studies have reported characteristic epidemiological pattern of 2022-mpox outbreak distinguished from sporadic cases and local outbreaks reported during the last 50 years in African countries.25][26][27][28][29] Further, the age of the infected was also distinct from pre-2022 outbreaks and cases.Most of the cases were reported in people aged between 30 and 40 years, which is a new characteristic of 2022 outbreak.0 The majority of the deaths associated with monkeypox virus were also reported among children aged below 10 years before 2010.However, after 2010 the death rate has shifted swiftly to people aged greater than 20 years.This study suggests that epidemiological changes in mpox outbreaks have been ongoing with consistent changes since 2010.However, majority of the studies on pre-2022 outbreaks have documented animals as the primary source of monkeypox virus transmission and human-to-human cases were rare.
During the 2022-outbreak we detect that majority of the cases (100%) ,21,24,29 These epidemiological changes are probably the result of genetic changes of the virus and origin of strains with altered properties and changes in human behaviors.Among the methods of transmission, direct contact with lesions, body fluids (pus, blood, tissue secretion, droplets of mouth or nose, semen), nonliving objects contaminated with monkeypox virus and droplets contributed to the majority of the cases. 8,10,12,15,18,21,24,27ecifically, MSM got infected from their partner through direct contact during sexual activities.Majority of the cases (70%-98%) were reported by MSM men during 2022-mpox outbreak.[47][48][49] Another major characteristic of this outbreak was the presence of previous sexually transmitted diseases, including HIV, syphilis, and gonorrhea among the monkeypox virus cases.About 30%-50% of the cases had previous records of HIV-positive health conditions.
,21,24,29 However, studies on the correlation between the presence of HIV and other STIs with monkeypox virus are limited and fail to determine their health impact.
Characteristic clinical presentations of monkeypox virus cases in 2022 outbreak were different from previous outbreaks and sporadic cases.16]18,21,24,29 Further, report of fever (60%-85%), weakness (30%-50%), pharyngitis (15%-40%), and proctitis isolates from lineage IIa were capable of infecting a large number of wild animals, laboratory animals, and from animal to humans. 4,46ough the natural history of monkeypox virus is not fully known, it is predicted that without the animal host, monkeypox virus cannot continue its infection cycle for a longer period in humans alone.However, isolates from IIb have several distinct epidemiological and clinical characteristics.][26][27][28][29]50,[56][57][58][59][60] For treatment and prevention, no drug or vaccine is approved against monkeypox virus.However, vaccination against smallpox has a good protective effect against monkeypox virus and can be used in specific situations.
The main strength of this study is the using of bias free approach to integrate the findings.This study included well-defined terms and broad search strategy on mpox without the limit of language, time, and place.In addition, a wide source of information, including gray literature, was also analyzed.However, this study has few limitations.
A lower number of actual cases may be presented due to lack of active and proper surveillance.Lack of studies on asymptomatic cases might have influenced the transmission characterization.

| CONCLUSION
The reduction of immunity against smallpox due to discontinuation of vaccination has contributed to the reappearance of monkeypox virus.
Evolution of lineage IIb with great capacity to spread from human-tohuman has impact on the larger outbreak of mpox in the nonendemic regions.We documented distinct epidemiological, clinical, and transmission properties of monkeypox virus during 2022 outbreak.
This study reported that men aged 30-40 years with specific sexual behavior (bisexual or MSM) contributed to majority of the infected people.Furthermore, household transmission and human-to-human transmission were two main sources of community outbreak.We also detected that distinct clinical feature included appearance of lesions on the genitals, anogenital, perianal, and trunk were common.This study provided an integrated insight into the epidemiology, clinical features, evolution, and transmission of monkeypox virus.

(
30 of 1400), respectively (Figure2B).The tree was built by using the maximum composite likelihood model and MPXV-M5312_HM12_Rivers was used as the reference sequence.From the phylogenetic tree we can see that isolates from lineage B.1 and subsequent lineages were more common in Europe, North America, and South America during 2017 to March 2023.After the 2022mpox outbreak, the spread of isolates from lineage B.1 and related lineages including B.1.2,B.1.10,B.1.1, and B.1.3occurred rapidly in Europe and North America (Figure 2C,D).These data may represent partial diversity as the sequencing of large number of cases is ongoing.Recently evolving lineages of hmpxv1 have an estimated evolutionary rate of ~6 × 10 −5 subs per site per year.The highest divergence that was calculated for the circulation lineages was 0.00035.The ongoing pandemic isolates of B.1 lineage and associated segregated lineages have a strong evolutionary relationship with the isolates of 2017-2019 outbreaks in evidence of animal-to-human and human-to-human transmission increased in Africa.Close contact with the lesions and body fluids of diseased animals during different activities like deforestation, hunting, slaughtering, and butchering contributed to interspecies transmission of animal-to-human infection.Further, animals like rodents get infected when they come in contact with lesions or body F I G U R E 2 (A) Phylogenetic tree of monkeypox virus, (B) distribution of different lineages of hmpxv1 across different continents, (C) divergence of hmpxv1 during the 2022 outbreaks, (D) proportionate distribution of different lineages of hmpxv1 in different regions during the 2022 outbreaks.The trees were built by the maximum composite likelihood method.Reference sequence MPXV-M5312_HM12_Rivers was used.Data were retrieved from NCBI and trees were adopted from Nextstrain. 54,55fluids of diseased humans.However, virological confirmation is lacking in Central and West African countries on the interspecies transmission of mpox virus.The cessation of smallpox vaccination since 1980s contributed to the wanning of cross-protection against monkeypox virus. 4Humanto-human transmission of monkeypox virus has been reported in both Central and West African countries. 4,48,50,56-60Contact with skin lesions and fluids of indexed patients among the family members and healthcare providers was the most attributed risk of transmission of monkeypox virus.Contaminated fomites, bedding, and clothing also contributed to the transmission. 4,13,15,18,56,57,59,60Sexual transmission of monkeypox virus before the 2022 outbreak was not prominent.Human-to-human transmission in both clade I and clade II was documented before the 2022-mpox pandemic in the endemic Majority of the studies reported evidence of sexual activities for transmission of monkeypox virus in non-endemic regions in 2022.The clinical, epidemiological, and virological findings strongly support the sexual transmission of monkeypox virus from indexed patients to susceptible persons.Direct contact with the lesions especially on the skin of the genital, anus, anorectum, and throat of patients during sexual activities contributed to the transmission of monkeypox virus (60-100%) monkeys, apes, rodents, shrews, pigs, small mammals, and dogs.50,[62][63][64][65][66]Nonhuman primates in both the wild environment including chimpanzees (Pan troglodytes) and monkeys of genera Cercopithecus and captivities including captive gorillas (Gorilla gorilla), gibbons (Hylobates lar), Asian orangutans (Pongo pygmaeus), chimpanzees, marmosets (Hapale jacchus), Siamiri and Macaca have been documented with clinical infection of mpox.

(
20%-40%) were more common during 2022 outbreak than pre-2022 outbreaks.Majority of the cases were reported in the outpatient ward in this outbreak, which was inpatient for pre-2022 outbreaks.This study documented that monkeypox virus from clades I, IIa, and IIb were involved in previous outbreaks depending on the geographic regions.However, during the 2022 outbreak, 100% of the isolates were from clade IIb with divergence of 0.0018-0.0035.About 26 lineages of clade IIb evolved and transmitted during 2022 outbreak.In the United States and Europe, isolates from B.1 lineage were the most predominant, followed by B.1.2 and B.1.10.Monkeypox virus variants from clade IIb were involved in the majority of the cases and responsible for distinct epidemiological features from pre-2022 outbreaks.As more human-to-human transmission occur the changes in the genome accelerated. 4,53-57Lineage I and IIa were confined to the majority of the cases in Central Africa and West Africa, respectively.However, emergence of the new West African lineage, IIb, was involved with large number of cases outside Africa and in 2022 outbreak.Before 2022 outbreak, Selection procedure of peerreviewed articles.The excluded articles were duplicate, reviews, correspondence, editorial, and failed to meet inclusion criteria.Epidemiological characteristics of patients with monkeypox virus infection.
T A B L E 1 (Continued) Abbreviations: IQR, interquartile range; MSM, men who have sex with men; STI, sexually transmitted infection.a Includes perianal skin.b Either skin, anogenital, or oropharyngeal samples combined.c Argentina, Belgium, Canada, Denmark, France, Germany, Israel, Portugal, South Africa, Spain, Sweden, Romania, The Netherlands, United Kingdom, and United States.d Range in years.e Argentina, Australia, Belgium, Canada, Denmark, France, Germany, Israel, Italy, Mexico, Portugal, Spain, Switzerland, The Netherlands, United Kingdom, and United States.f Skin or anogenital samples combined.g Mean age.
T A B L E 5 b Argentina,